Homogeneous detection method

ABSTRACT

The invention relates to methods for the quantitative or qualitative detection of an analyte in an assay and adequate reagents therefor, particularly a homogeneous binding test. According to the invention, an analyte-specific binding partner R1 comprises more than one specific binding point for a specific binding partner X that is associated with a component of a signal-forming system while a second analyte-specific binding partner R2 comprises more than one specific binding point for a specific binding partner Y which is also associated with a component of a signal-forming system.

This is a continuation of PCT International Application No.PCT/EP2004/012648, filed Nov. 9, 2004, which is a continuationapplication of U.S. patent application Ser. No. 10/725,070, filed Dec.1, 2003, now abandoned, and claims the benefit of U.S. ProvisionalApplication No. 60/526,116, filed Dec. 1, 2003, all of which areincorporated herein by reference.

The invention relates to methods for quantitatively or qualitativelydetecting an analyte in a sample and to suitable reagents for thispurpose.

For the purpose of detecting analytes, use is frequently made of bindingtests in which the specific binding of analyte to be detected toanalyte-specific binding partners makes it possible to draw conclusionsas to the presence, absence or quantity of the analyte in a sample.Immunoassays or methods in which oligonucleotides or polynucleotides arehybridized are examples of binding tests.

What are termed the “heterogeneous binding tests” are characterized byone or more separation steps and/or washing steps. The separation can beeffected, for example, by immunoprecipitation, precipitation withsubstances such as polyethylene glycol or ammonium sulfate, filtration,magnetic separation or binding to a solid phase. Such a “solid phase”consists of porous and/or nonporous material which is as a ruleinsoluble in water. It can have a very wide variety of forms such as: avessel, a small tube, a microtitration plate, a sphere, a microparticle,a rod or a strip, or filter paper or chromatography paper, etc. In thecase of heterogeneous binding tests in sandwich format, one of theanalyte-specific binding partners is as a rule bound to a solid phaseand is used for separating off the “analyte/analyte-specific bindingpartner” binding complex from the liquid phase while the otheranalyte-specific binding partner carries a detectable label (e.g. anenzyme, a fluorescent label or a chemiluminescent label, etc.) for thepurpose of detecting the binding complex. These test methods are furthersubdivided into what are termed single-step sandwich tests, in which thetwo specific binding partners are incubated simultaneously with thesample, and into two-step sandwich tests, in which the sample is firstof all incubated with the solid phase reagent and, after a separationand washing step, the solid phase-bound binding complex, consisting ofanalyte and analyte-specific binding partner, is incubated with thedetection reagent.

In “homogeneous binding tests”, no separation takes place between freecomponents of the signal-generating system and components of the systemwhich are bound to the “analyte/analyte-specific binding partner”complex. The test mixture, which contains the analyte-specific bindingpartners, the signal-forming components and the sample, is measuredafter, or even during, the binding reaction without any furtherseparation and/or washing step and the corresponding measurement signalis determined. Examples of homogeneous immunoassays (see also Boguslaski& Li (1982) Applied Biochemistry and Biotechnology, 7: 401-414) are manyturbidimetric or nephelometric methods, with it being possible for theanalyte-specific binding partners, which are used for the detection, tobe associated with latex particles; EMIT® tests; CEDIA® tests;Fluorescent-Polarization Immunoassays; Luminescent Oxygen ChannelingImmunoassays (“LOCI”, see EP-A2-0 515 194; Ullman et al. (1994) Proc.Natl. Acad. Sci., 91: 5426-5430; Ullman et al., (1996) ClinicalChemistry, 42: 1518-1526); etc. In a homogeneous sandwich immunoassay,such as a nephelometric latex test, the antibody reagents are incubatedtogether with the sample and the signal is measured during and/or afterthe incubation without any separation or washing step being carried outprior to the measurement. Expressed in other words: the antibody-boundanalyte is not separated from the free analyte or from antibodies whichhave not bound any analyte.

Homogeneous and heterogeneous binding tests can also be carried out inthe form of what is termed a “sandwich assay”. In this case, the analyteis, for example in a heterogeneous binding test, bound by a solidphase-associated analyte-specific binding partner and ananalyte-specific binding partner which is associated with a component ofa signal-generating system. In sandwich immunoassays, antibodies orantigens or haptens can be the analyte-specific binding partners.

The “indirect immunoassay” is another special embodiment of aheterogeneous or homogeneous binding test. In this case, the analyte isan antibody. One of the analyte-specific binding partners is theantigen, or a modified antigen, of the antibody (=analyte) to bedetected and the other analyte-specific binding partner is as a rule animmunoglobulin-binding protein, such as an antibody which is able tospecifically bind the antibody (=analyte) to be detected.

In a homogeneous or heterogeneous “competitive binding test”, sampleanalyte and reagent analyte (for example a “modified analyte” such as alabeled analyte, analyte fragment or analyte analog) compete for bindingto a limited number of analyte-specific binding partners. Examples forillustrating the principle: (i) sample analyte competes with reagentanalyte, which is associated with a component of a signal-generatingsystem, for binding to solid phase-associated analyte-specific bindingpartners or (ii) sample analyte competes with solid phase-associatedanalyte (=reagent analyte) for binding to analyte-specific bindingpartners which are associated with a component of a signal-generatingsystem.

However, in the case of many binding tests, great difficulties arisewhen preparing the reagents since the binding of the analyte-specificbinding partners to solid phases or particulate components of asignal-generating system (e.g. microparticles) frequently causes theanalyte-specific binding partners which have been bound in this way toloose activity and/or gives rise to changes in the properties (e.g. asregards conformation or stability) of these binding partners. Thisapplies, in particular, when the analyte-specific binding partners whichhave been bound are proteins, such as antibodies or enzymes.

Attempts have therefore been made to remedy this problem by introducingwhat are termed “universal reagents” (see, e.g., EP-0 105 714). Thus,according to EP-0 245 926, it is possible to detect an analyte by using,as a universal solid-phase reagent, an avidin-coated solid phase towhich the biotinylated analyte-specific binding partner is bound. Othermethods use a biotinylated analyte-specific binding partner which isable, for example, to bind to a streptavidin/enzyme complex which isused as a universal detection reagent. However, in these tests,separation steps, such as washing steps, are an essential element incarrying out the test.

Particular difficulties in regard to using universal reagents arise inhomogeneous binding tests, particularly in homogeneous binding testswhich are based on using particulate universal reagents (e.g.streptavidin-coated microparticles). If the analyte-specific bindingpartner were divalent or polyvalent, i.e. possessed two or more bindingsites for the particulate universal reagents (e.g. if it were anantibody to which two or more biotin molecules were bound), this wouldthen lead to the microparticle being agglutinated even without ananalyte being present in the sample. This would then result in erroneousdeterminations. According to EP-0 356 964, EP-0 349 988 and EP 0 444561, it is therefore regarded as being essential for being able to carryout such a homogeneous test that the analyte-specific binding partner ismonovalent in regard to the universal reagent, i.e. that the bindingpartner only possesses one binding site for the universal reagent (e.g.streptavidin-latex particle). In the case of homogeneous LOCI tests aswell (see EP-0 515 194), the inventors point out that the generation ofa measurement signal depends on the formation of particle pairs which ineach case consist of one sensitizer particle and one chemiluminescerparticle (Ullman et al., (1996) Clinical Chemistry, 42: 1518-1526). Adisadvantage of these methods is that the number of binding sites on thecorresponding analyte-specific binding partner has to be controlledprecisely.

EP-0 138 297 takes another approach. In this case, the number ofbiotinylated antibodies which are to bind to avidin-coated latexparticles is controlled by it being necessary to add free biotin.However, a measure of this nature has a negative effect both on reagentstability and on the analytical sensitivity of the test. Furthermore,the universal reagent in this case reacts with the correspondinganalyte-specific binding partner prior to the actual test method, i.e.the antibodies which are bound to the latex particles by way of abiotin/avidin bridge constitute the reagent which is to be employed inthe test. This suffers from the disadvantage that the universal reagentcannot be used on the analytical unit with different analyte-specificbinding partners depending on the test which is to be carried out.

The object was therefore to develop an improved method for detecting ananalyte, in particular using homogeneous test procedures, with thismethod not exhibiting the above-described disadvantages. A method ofthis nature can be used particularly advantageously in automatedanalytical equipment.

This object is achieved by providing the novel method and materialswhich are described in the claims.

The novel method achieves this object by making available universalreagents (specific binding partners X or Y, each of which is associatedwith a component of a signal-generating system), which can be adjustedindependently of the analyte-specific reagents (analyte-specific bindingpartners R1 and R2) to the specific interests, and enabling analytes tobe detected with a high degree of sensitivity and precision.

Since the analyte-specific binding partners of the signal-generatingcomponents can be used independently of each other and since theanalyte-specific binding partners and the signal-generating componentscan be optimally adjusted, independently of each other, to the givenrequirements, this invention also solves the following general problemof homogeneous binding tests, namely the mutually opposed requirementsfor optimal differentiation and optimal sensitivity: the concentrationof the reagents should, on the one hand, be limited so as to ensure thatthe background signals are as low as possible and, on the other hand,the reagents should be highly concentrated and highly labeled in orderto achieve a satisfactory change in signal per unit of time.

The analyte-specific, novel binding partners R1 and/or R2 arecharacterized by the fact that they exhibit more than one binding sitefor the respective specific binding partner X or Y which is associatedwith components of a signal-generating system. Preference is given, inthe novel method, to using universal reagents which comprise componentsof a signal-generating system which can interact with each other, e.g.in the form of an energy transfer, over very short distances.

It has been found, surprisingly, that the novel use, in homogeneous testmethods, of analyte-specific binding partners which possess more thanone binding site for the given universal reagent does not lead toerroneous measurements but, on the contrary, to what is even animprovement in the ratio of background signal to analyte-specificmeasurement signal (see, e.g., Tables 3 and 4).

Since the binding sites of the analyte-specific binding partners for thegiven universal reagents preferably consist of small molecules, e.g.haptens such as digoxigenin, biotin, DNP or FITC, which are preferablybonded covalently to the analyte-specific binding partners, theanalyte-specific activity or binding capacity of R1 or R2 is as a rulenot impaired or hardly impaired. The binding sites can also be part ofthe unaltered analyte-specific binding partner. Thus, theanalyte-specific binding partner could, for example, be a human IgGantibody which possesses several binding sites which are specificallyrecognized by anti-human IgG antibodies from another species.

The invention preferably relates, in one instance, to a homogeneousmethod for quantitatively or qualitatively detecting an analyte in asample, with the analyte-specific binding partner R1 possessing specificbinding sites for the specific binding partner X, which is associatedwith a component of a signal-generating system, and the analyte-specificbinding partner R2 possessing specific binding sites for the specificbinding partner Y, which is associated with a component of asignal-generating system, which comprises R1 and/or R2 possessing morethan one binding site for the respective specific binding partner whichis associated with components of a signal-generating system. Aparticular advantage of the invention is that the specific bindingpartners X and/or Y (e.g. avidin, streptavidin, etc.) do not, asdescribed in EP-0 138 297, have to be saturated by adding free “specificbinding sites” (e.g. biotin).

This novel method is particularly preferably a homogeneous binding test,in particular a homogeneous immunoassay. As already explained above,this homogeneous binding test can be carried out, inter alia, in theform of a sandwich assay, an indirect immunoassay or a competitivebinding test.

Some terms which have been used for describing the invention areexplained in more detail below:

A “quantitative detection” measures the quantity, concentration oractivity of the analyte in the sample. The term “quantitative detection”also encompasses semiquantitative methods which only record theapproximate quantity, concentration or activity of the analyte in thesample or can only be used to give an indication of the relativequantity, concentration or activity. A “qualitative detection” is to beunderstood as meaning simply detecting whether the analyte or itsactivity is present or absent in the sample or indicating that thequantity, concentration or activity of the analyte in the sample isbelow or above one specific threshold value or several specificthreshold values.

The term “analyte” is to be understood as meaning the substance which isto be detected in the novel method. Examples of an analyte are listed onpages 8-15 in EP-A2-0 515 194. The analyte can be a member of a specificbinding pair. The analyte may possess one binding site (monovalent,usually a hapten) or several binding sites (polyvalent). Inimmunochemical tests, such a binding site is frequently also termed anepitope. In addition, the analyte can be a single substance or a groupof substances which possess at least one single shared binding site.

A monovalent analyte generally has a molecular weight of from about 100to 2000, in particular of from 125 to 1000. Many oligopeptides,oligonucleotides, oligosaccharides, pharmaceuticals, drugs, metabolites,pesticides, etc. are covered by the term monovalent analyte. Apolyvalent analyte generally has a molecular weight of more than 2000,usually more than 10,000. Examples of polyvalent analytes arepolypeptides, polysaccharides, nucleic acids, cells, cell constituents,including chromosomes, genes, mitochondria and other cell organelles,cell membranes, etc. Proteins are frequently the substances which are tobe detected. These proteins may be members of a protein family which arecharacterized by similar structural features and/or a similar biologicalfunction. Examples of analytically interesting protein families arepathogen proteins, immunoglobulins, cytokines, enzymes, hormones, tumormarkers, metabolic markers, tissue-specific antigens, histones,albumins, globulins, scleroproteins, phospho-proteins, mucines,chromoproteins, lipoproteins, nucleoproteins, glycoproteins,proteoglycans, receptors, HLA, coagulation factors, cardiac infarctionmarkers (e.g. myoglobin, troponin, pro-BNP, etc.), etc. Examples ofother analytically interesting substances are single-stranded ordouble-stranded oligonucleotides and polynucleotides.

Within the meaning of the invention, a “sample” is to be understood asbeing the material which is suspected of containing the substance(“analyte”) to be detected. For example, the term sample encompassesbiological fluids or tissue which is derived, in particular, from humansor animals, such as blood, plasma, serum, sputum, exudate,bronchoalveolar lavage, lymph fluid, synovial fluid, seminal fluid,vaginal mucus, feces, urine, spinal fluid, hair, skin and tissue samplesor tissue sections. The term also comprises cell culture samples, plantfluids or tissues, forensic samples, water and sewage samples,foodstuffs and pharmaceuticals.

In addition, the term “sample” also encompasses a pretreated samplewhich may contain the substance (“analyte”) to be detected in a form inwhich it is released from carrier substances or is amplified: a numberof samples have to be pretreated in order to make the analyte availablefor the detection method or in order to remove sample constituents whichinterfere. Such pretreatment of samples may involve the separationand/or lysis of cells, the precipitation, the hydrolysis or thedenaturation of sample constituents such as proteins, centrifugation ofsamples, treatment of the sample with organic solvents such as alcohols,in particular methanol; or treatment of the sample with detergents. Thesample is frequently transferred into another, usually aqueous, mediumwhich is intended to interfere as little as possible with the detectionmethod. The analyte may also be amplified. Amplification of nucleicacids leads, for example, to the generation of one or more copies of thenucleic acid chain to be detected. Such amplification methods, e.g. thepolymerase chain reaction (PCR), the ligase chain reaction (LCR),amplification using Q beta replicase, nucleic acid sequence-basedamplification (NASBA), single primer amplification (ASPP), and others,are well known to the skilled person.

An “analyte-specific binding partner” is to be understood as beingeither a specific binding partner which is able to bind specifically tothe analyte or a specific binding partner (e.g. a modified analyte)which is able to bind to another analyte-specific binding partner. As arule, a “modified analyte” is a substance which is at least able to bindto an analyte-specific binding partner but which differs from the sampleanalyte in lacking, or possessing additional, binding sites, e.g. abiotinylated analyte or an analyte which is associated with a componentof a signal-generating system. A modified analyte is used, for example,in competitive tests.

A “specific binding partner” is to be understood as being a member of aspecific binding pair. The members of a specific binding pair are twomolecules each of which possesses at least one structure which iscomplementary to a structure possessed by the other molecule, with thetwo molecules being able to bind together specifically by way of a bondbetween the complementary structures. In this connection, the termmolecule also encompasses molecular complexes such as enzymes whichconsist of an apoenzyme and a coenzyme, proteins which consist ofseveral subunits, lipoproteins which consist of protein and lipids, etc.Specific binding partners can be naturally occurring substances or elsesubstances which are prepared, for example, by means of chemicalsynthesis, microbiological techniques and/or recombinant DNA methods.Thus, it is by now possible to select specific binding partners usingphage display libraries, synthetic peptide databases or recombinatorialantibody libraries (Larrick & Fry (1991) Human Antibodies andHybridomas, 2: 172-189). The following may be mentioned as examples forthe purpose of illustrating the term specific binding partner, withoutthis being understood as any restriction: thyroxin-binding globulin,steroid-binding proteins, antibodies, antigens, haptens, enzymes,lectins, nucleic acids, repressors, oligonucleotides, polynucleotides,protein A, protein G, avidin, streptavidin, biotin, complement componentC1q, nucleic acid-binding proteins, etc. Examples of specific bindingpairs are: antibody-antigen, antibody-hapten, digoxigen/anti-digoxigenantibody, fluorescein/anti-fluorescein antibody, operator-repressor,nuclease-nucleotide, biotin-avidin, biotin/streptavidin,lectin-polysaccharide, steroid-steroid-binding protein, activecompound-active compound receptor, hormone-hormone receptor,enzyme-substrate, IgG-protein A, complementary oligonucleotides orpolynucleotides, etc. In what are termed homogeneous gene probe tests,the specific binding partners are as a rule nucleic acid chains whichare at least in part complementary to segments of the nucleic acid chainwhich is to be detected.

Within the meaning of this invention, the term “antibody” is to beunderstood as signifying an immunoglobulin, e.g. an immunoglobulin ofthe class or subclass IgA, IgD, IgE, IgG₁, IgG_(2a), IgG_(2b), IgG₃,IgG₄ or IgM. An antibody possesses at least one binding site (frequentlytermed a paratope) for an epitope (frequently also termed antigenicdeterminant) on an antigen or hapten. Such an epitope is characterized,for example, by its spatial structure and/or by the presence of polarand/or apolar groups. The binding site possessed by the antibody iscomplementary to the epitope. The antigen-antibody reaction or thehapten-antibody reaction functions in accordance with what is termed the“key-lock principle” and is as a rule highly specific, i.e. theantibodies are able to distinguish between slight differences in theprimary structure, in the charge, in the spatial configuration and inthe steric arrangement of the antigen or hapten. What are termed thecomplementarity determining regions possessed by the antibody make aparticular contribution to binding the antibody to the antigen orhapten.

The term “antigens” encompasses monovalent and polyvalent antigens. Apolyvalent antigen is a molecule or a molecule complex to which morethan one immunoglobulin can bind simultaneously whereas only one singleantibody can bind at any one time to a monovalent antigen. A moleculewhich is not immunogenic on its own, but which is usually bound to acarrier for immunization purposes, is generally termed a hapten.

Within the meaning of this invention, the term antibody is not only tobe understood as signifying complete antibodies but also, expressly,antibody fragments such as Fab, Fv, F(ab′)₂ and Fab′; and also chimeric,humanized, bispecific, oligospecific or single-chain antibodies; and,furthermore, also aggregates, polymers and conjugates of immunoglobulinsand/or their fragments provided the properties of binding to antigen orhapten are retained. Antibody fragments can be prepared, for example, byenzymically cleaving antibodies using enzymes such as pepsin or papain.Antibody aggregates, antibody polymers and antibody conjugates can begenerated using a wide variety of methods, e.g. by heat treatment, byreaction with substances such as glutaraldehyde, by reaction withimmunoglobulin-binding molecules, by biotinylating antibodies andsubsequently reacting them with streptavidin or avidin, etc.

Within the meaning of this invention, an antibody can be a monoclonalantibody or a polyclonal antibody. The antibody can have been preparedusing the customary methods, e.g. by immunizing the human or an animal,such as a mouse, rat, guinea pig, rabbit, camel, horse, sheep, goat orchick (see also Messerschm id (1996) BIOforum, 11: 500-502), andsubsequently isolating antiserum; or else by establishing hybridomacells and subsequently purifying the secreted antibodies; or else bycloning and expressing the nucleotide sequences, or modified versionsthereof, which encode the amino acid sequences which are responsible forbinding the natural antibody to the antigen and/or hapten. RecombinantDNA methods can also be used, where appropriate, to prepare antibodiesin plant, such as yeast cells (Fischer et al. (1999) Biol. Chem., 380:825-839; Hiatt et. al. (1992) Genetic Engineering, 14, 49-64)), animalcells, prokaryotic cells (see, e.g., WO 95/25172) and isolated humancells.

A “signal-generating system” can consist of one or more components, withat least one of the components being a detectable label. A label is tobe understood as being any molecule which itself produces a signal orwhich can induce the production of a signal, such as a fluorescentsubstance, a radioactive substance, an enzyme or a chemiluminescentsubstance. The signal can be detected or measured, for example, usingthe enzyme activity, the luminescence, the light absorption, the lightscattering, the emitted electromagnetic or radioactive radiation, or achemical reaction.

A “label” is able itself to generate a detectable signal such that nofurther components are required. Many organic molecules absorbultraviolet and visible light, with these molecules being able to comeinto an excited energy state, as a result of the energy transferred bythe absorption of the light, and emitting the absorbed energy in theform of light of a wavelength which is different from that of theincident light. Yet again other labels, such as radioactive isotopes,dyes or magnetic and nonmagnetic microparticles, are able to directlygenerate a detectable signal.

Yet again other labels require further components in order to generatethe signal, i.e., in such a case, the signal-producing system includesall the components, such as substrates, coenzymes, quenchers,accelerators, additional enzymes, substances which react with enzymeproducts, catalysts, activators, cofactors, inhibitors, ions, etc.,which are required for generating a signal.

Examples of suitable labels (see also EP-A2-0 515 194; U.S. Pat. No.5,340,716; U.S. Pat. No. 5,545,834; Bailey et al. (1987) J.Pharmaceutical & Biomedical Analysis 5: 649-658) are enzymes, includinghorse radish peroxidase, alkaline phosphatase, glucose-6-phosphatedehydrogenase, alcohol dehydrogenase, glucose oxidase, β-galactosidase,luciferase, urease and acetylcholinesterase; dyes; fluorescentsubstances, including fluorescein isothio-cyanate, rhodamine,phycoerythrin, phycocyanin, ethidium bromide,5-dimethylaminonaphthalene-1-sulfonyl chloride and fluorescent chelatesof rare earths; chemiluminescent substances, including luminol,isoluminol, acridinium compounds, olefin, enol ethers, enamine,arylvinyl ethers, dioxene, arylimidazole, lucigenin, luciferin andaequorin; sensitizers, including eosin, 9.10-dibromoanthracene,methylene blue, porphyrin, phthalocyanin, chlorophyll and rose Bengal;coenzymes; enzyme substrates; radioactive isotopes, including ¹²⁵I,¹³¹I, ¹⁴C, ³H, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁹Fe, ⁵⁷Co and ⁷⁵Se; particles,including magnetic particles or particles, preferably latex particles,which can themselves be labeled, for example with dyes, sensitizers,fluorescent substances, chemiluminescent substances, isotopes or otherdetectable labels; sol particles, including gold sols or silver sols;liposomes or cells which can themselves be labeled with detectablelabels; etc.

A signal-generating system can also comprise components which, whenspatially close to each other, are able to enter into a detectableinteraction, for example in the form of energy donors and energyrecipients such as photosensitizers and chemiluminescent substances(EP-A2-0 515 194), photosensitizers and fluorophores (WO 95/06877),radioactive iodine¹²⁵ and fluorophores (Udenfriend et al. (1985) Proc.Natl. Acad. Sci. 82: 8672-8676), fluorophores and fluorophores (Mathis(1993) Clin. Chem. 39: 1953-1959) or fluorophores and fluorescentquenchers (U.S. Pat. No. 3,996,345).

An interaction between the components includes the direct transfer ofenergy between the components, for example as the result of light orelectron radiation and also by way of short-lived reactive chemicalmolecules. In addition, it also includes processes in which the activityof one component is inhibited or augmented by one or more othercomponents, for example an inhibition in or increase of the enzymeactivity or an inhibition, increase or change (e.g. wavelength shift,polarization) in/of the electromagnetic radiation which is emitted bythe affected component. Interaction between the components also includesenzyme cascades. In this case, the components are enzymes, at least oneof which supplies the substrate for another enzyme, thereby resulting inthe coupled substrate reaction having a maximal or minimal rate.

As a rule, an efficient interaction between the components takes placewhen these components are spatially adjacent, that is, for example,within a distance range of a few μm, in particular within a distancerange of less than 600 nm, preferably less than 400 nm, veryparticularly preferably less than 200 nm.

In a very particularly preferred method according to the invention, thesignal-generating system comprises microparticle-associatedphotosensitizers and microparticle-associated chemiluminescentsubstances.

Microparticles are frequently used as a solid phase and/or as a label.Within the meaning of this invention, the term “microparticles” is to beunderstood as signifying particles which have an approximate diameter ofat least 20 nm and not more than 20 μm, generally between 40 nm and 10μm, preferably between 0.1 and 10 μm, particularly preferably between0.1 and 5 μm, very particularly preferably between 0.15 and 2 μm. Themicroparticles can have a regular or irregular shape. They can bespheres, spheroids or spheres possessing cavities or pores of greater orlesser size. The microparticles can consist of organic material orinorganic material or of a mixture or combination of both materials.They can consist of a porous or nonporous material and of a swellable ornonswellable material. While the microparticles can in principle be ofany density, preference is given to particles which are of a densitywhich approximates that of water, such as from about 0.7 to about 1.5g/ml. The preferred microparticles can be suspended in aqueous solutionsand are stable in suspension for as long as possible. They may betransparent, partially transparent or nontransparent. The microparticlescan consist of several layers, such as what are termed core and shellparticles, having a core and one or more enveloping layers. The termmicroparticles encompasses, for example, dye crystals, metal sols,silica particles, glass particles, magnetic particles, polymerparticles, oil drops, lipid particles, dextran and protein aggregates.Preferred microparticles are particles which can be suspended in aqueoussolutions and which consist of water-insoluble polymer material,particularly substituted polyethylenes. Very particular preference isgiven to latex particles, for example composed of polystyrene, acrylicacid polymers, methacrylic acid polymers, acrylonitrile polymers,acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate,polyvinylpyridine or vinyl chloride-acrylate. Latex particles whichposses reactive groups, such as carboxyl, amino or aldehyde groups, ontheir surface, with these groups enabling specific binding partners, forexample, to bind covalently to the latex particles, are of particularinterest. The preparation of latex particles is described, for example,in EP 0 080 614, EP 0 227 054 and EP 0 246 446.

The term “associated” is to be understood broadly and comprises, forexample, a covalent bond and a noncovalent bond, a direct bond and anindirect bond, absorption to a surface and enclosure in an invaginationor a cavity, etc. In the case of a covalent bond, the specific bindingpartner is, for example, bonded to a label by way of a chemical bond. Acovalent bond is usually said to exist between two molecules when atleast one atomic nucleus in the first molecule shares electrons with atleast one atomic nucleus in the second molecule. Examples of anoncovalent bond are surface adsorption, enclosure in cavities or thebinding of two specific binding partners. In addition to direct bindingto a label, the specific binding partners can also be bonded to thelabel indirectly by way of specific interaction with other specificbinding partners. This will be illustrated by way of an example: abiotinylated anti-fluorescein antibody can be bound to the label by wayof label-bound avidin.

A microparticle can posses a coating consisting of one or more layers,for example composed of proteins, carbohydrates, biopolymers, organicpolymers, or mixtures thereof, in order, for example, to suppress orprevent the nonspecific binding of sample constituents to the particlesurface or in order, for example, to achieve improvements in regard tosuspension stability, stability during storage, conformational stabilityor resistance to UV light, microbes or other agents having a destructiveeffect. Thus, this coating can, in particular, consist of protein layersor polymer layers, such as cyclodextrins, dextrans, hydrogels, albuminor polyalbumins, which have been applied covalently or adsorptively tothe microparticles.

In the inventive method, R1 and/or R2 can be bound to components of thesignal-generating system by way of X and/or Y before, during or afterthe binding reaction with the analyte. The sample can be initiallyincubated with the analyte-specific binding partners R1 and R2, with thespecific binding partners X and Y then being added subsequently.However, the reagents can also be added in another sequence.

When implementing a particularly preferred embodiment of the novelhomogeneous binding test, the sample is first of all mixed sequentiallyor simultaneously with the analyte-specific binding partners R1 and R2and, after that, the components of the signal-generating system,together with the binding partners X and Y, are added sequentially orsimultaneously to the mixture.

The number of binding sites which the novel analyte-specific bindingpartner R1 possesses for the specific binding partner X should be atleast 2, preferably at least 5, particularly preferably at least 10 andvery particularly preferably at least 15, and the number of bindingsites which the novel analyte-specific binding partner R2 possesses forthe specific binding partner Y should be at least 2, preferably at least5, particularly preferably at least 10 and very particularly preferablyat least 15.

The novel analyte-specific binding partners R1 and R2 can also be oneand the same analyte-specific binding partner or be differentanalyte-specific binding partners. Thus, in a sandwich immunoassay, forexample, a monoclonal antibody can be used both as analyte-specificbinding partner R1 and as analyte-specific binding partner R2 if theanalyte possesses more than one epitope for this antibody.

In the novel method, the analyte-specific binding partners R1 and R2 areboth able, in the case of a sandwich assay or of an indirectimmunoassay, to bind the analyte specifically. In the case of a sandwichimmunoassay, for example, the analyte-specific binding partners can beanalyte-specific antibodies or, if the analyte is itself an antibody, beits antigen or a “modified antigen” or an antigen analog. In acompetitive test set-up, one of the novel analyte-specific bindingpartners R1 and R2 should be a modified analyte.

The binding sites which the novel analyte-specific binding partner R1possesses for the specific binding partner X are preferably haptens.Binding sites which are possessed by the novel analyte-specific bindingpartner R1 are particularly preferably biotin, digoxigenin, fluorescein,single-stranded nucleic acid chains or dinitrophenol. However, it isalso possible to use other molecules which are in each case a member ofa specific binding pair.

The binding sites which the novel analyte-specific binding partner R2possesses for the specific binding partner Y are preferably haptens. Thebinding sites possessed by the novel analyte-specific binding partner R2are particularly preferably biotin, digoxigenin, fluorescein,single-stranded nucleic acid chains or dinitrophenol. However, it isalso possible to use other molecules which are in each case a member ofa specific binding pair.

The novel specific binding partners X and Y can be one and the samespecific binding partner or different specific binding partners. Thenovel specific binding partner X is preferably avidin, streptavidin, ananti-digoxigenin antibody, an anti-dinitrophenol antibody, asingle-stranded nucleic acid chain or an anti-hapten antibody. However,it can also be an enzyme, an enzyme substrate or an antibody which isable to bind particular polypeptides, oligopeptides or enzymesspecifically. The novel specific binding partner Y can also be avidin,streptavidin, an anti-digoxigenin antibody, an anti-dinitrophenolantibody, a single-stranded nucleic acid chain or an anti-haptenantibody. However, it can also be an enzyme, an enzyme substrate or anantibody which is able to bind particular polypeptides, oligopeptides orenzymes specifically.

In a particularly preferred embodiment of the novel method, componentsof the signal-generating system are brought, as a result of the analytebeing bound to R1 and/or R2, to a distance from each other which permitsan interaction, in particular an energy transfer, between thesecomponents. The magnitude of this interaction is then measured for thepurpose of quantitatively or qualitatively detecting the analyte in thesample. This method is particularly suitable for sandwich assays andindirect immunoassays.

In another particularly preferred embodiment of the novel method,components of the signal-generating system are brought, as a result ofthe analyte being bound to R1 or R2, to a distance from each other whichpermits no interaction, or only a very slight interaction, in particularno energy transfer or only very slight energy transfer, between thesecomponents. The residual magnitude of this interaction is then measuredfor the purpose of quantitatively or qualitatively detecting the analytein the sample. This method is particularly suitable for competitivebinding tests.

In order to be able to increase the number of binding sites which theanalyte-specific binding partners possess for the specific bindingpartners X or Y, without decreasing the specificity or sensitivity ofthe analyte-specific binding partners, it is possible to introducecarrier molecules to which both the analyte-specific binding partnersand the binding sites can be bound. It is therefore advantageous, in thenovel method, if R1 is one or more analyte-specific binding partnerswhich is/are associated with a carrier molecule, with the carriermolecule being able to possess binding sites for the specific bindingpartner X. It is furthermore advantageous if R2 is also one or moreanalyte-specific binding partners which is/are associated with a carriermolecule, with the carrier molecule being able to possess binding sitesfor the specific binding partner Y.

In a particularly preferred embodiment according to the invention, R1and/or R2 is/are in each case associated with such a carrier molecule.Examples of suitable carrier molecules are proteins, for exampleantibodies, enzymes, albumins, such as bovine serum albumin or humanserum albumin, or protein polymers, dextrans, cyclodextrins, dendrimersor similar structures. Particularly preferred protein polymers orprotein aggregates can consist of antibodies, albumin molecules,enzymes, or mixtures thereof, which are associated with each other andare preferably covalently bonded. Very particular preference is given tousing biotinylated dextran and biotinylated protein polymers (e.g.biotinylated antibody polymers) as carrier molecules.

A particularly preferred carrier molecule can be prepared as describedin Example 7. In this case, murine antibodies, preferably murine IgGantibodies, are covalently bonded to each other using a couplingreagent. Carrier molecules according to the invention can also beprepared in an analogous manner from enzymes, antibodies (e.g. mouse orgoat antibodies, in particular IgG antibodies), albumins or mixturesthereof. The sites for binding the specific binding partner X or Y, e.g.biotin, digoxigenin, fluorescein, single-stranded nucleic acid chains,dinitrophenol, etc., can be bonded to these protein polymers usingmethods which are known to the skilled person, with a covalent bondbeing preferred.

An analyte-specific binding partner which is associated with a carriermolecule can, for example, be prepared as follows: the analyte-specificbinding partner, preferably an antibody, particularly preferably anantibody fragment, is bonded to the carrier molecule using methods whichare known to the skilled person, e.g. using coupling reagents. This bondshould if at all possible be covalent. A suitable carrier molecule is,for example, biotinylated dextran (see Example 5) or else one of theother carrier molecules described above, in particular those like theantibody polymers described in Example 7. As in the case of thebiotinylated dextran, the sites for binding the specific binding partnerX or Y can be introduced before or, as described in Example 7, after thebinding reaction between the analyte-specific binding partner and thecarrier molecule. Several analyte-specific binding partners, rather thanjust one, can also be bonded to a carrier molecule. However, in additionto this preferred embodiment according to the invention, it is alsopossible for several carrier molecules to be bonded to oneanalyte-specific binding partner.

Another part of the subject-matter of this invention is a carriermolecule which is associated with, preferably covalently bonded to, oneor more analyte-specific binding partners. In one embodiment of thissubject-matter, the carrier molecule is a protein polymer, for exampleantibodies (see Example 7), albumin molecules, enzymes, or mixturesthereof, which are covalently bonded to each other, which canadditionally possess binding sites, e.g. biotin, digoxigenin,fluorescein, single-stranded nucleic acid chains, dinitrophenol, etc.,for a specific binding partner X or Y. The number of the binding sitesshould be at least 2, still better more than 5, preferably more than 10,particularly preferably more than 15, very particularly preferably morethan 18, per carrier molecule, which is associated with one or moreanalyte-specific binding partners. In this embodiment according to theinvention, the analyte-specific binding partner is preferably anantibody or an antibody fragment, an antigen, a hapten or a nucleic acidchain.

The use of the above-described carrier molecule, which is associatedwith one or more analyte-specific binding partners, in a homogeneous orheterogeneous binding test for the purpose of quantitatively orqualitatively detecting an analyte in a sample, in particular in ahomogeneous or heterogeneous immunoassay, is also in accordance with theinvention.

In that which follows, analyte-specific binding partners which areassociated with the carrier molecules according to the invention arealso termed conjugates. These conjugates can naturally also be usedadvantageously in heterogeneous binding tests as well as in homogeneousbinding tests.

A conjugate according to the invention consists of a carrier moleculewhich is associated with one or more analyte-specific binding partners,with this conjugate possessing additional binding sites for a specificbinding partner X or Y. In a special embodiment of a conjugate, thecarrier molecule consists of dextran, cyclodextrin or dendrimers or ofantibodies which are bonded together, albumin molecules which are bondedtogether, enzymes which are bonded together, or mixtures thereof whichare bonded together. Their bond should preferably be covalent. Theadditional binding sites possessed by the conjugate according to theinvention can be biotin, digoxigenin, fluorescein, dinitrophenol orsingle-stranded nucleic acid chains. In a particularly preferredembodiment of the conjugate according to the invention, the carriermolecule is covalently bonded to one or more analyte-specific bindingpartners. The conjugate according to the invention should possess atleast 2, preferably more than 5, particularly preferably more than 10,very particularly preferably more than 15 and optimally more than 18additional binding sites for the specific binding partner X or Y. Veryparticular preference is given to a conjugate in which the carriermolecule consists of antibodies, preferably mouse or goat IgGantibodies, which are covalently bonded together. The invention alsorelates to a reagent which contains one or more of these conjugates andto a test kit which contains such a reagent.

The conjugates according to the invention can be used in a homogeneousor heterogeneous binding test (e.g. an immunoassay) for the purpose ofquantitatively or qualitatively detecting an analyte in a sample. In oneembodiment of a binding test according to the invention, in particular ahomogeneous binding test, a conjugate according to the invention whichpossesses specific binding sites for the specific binding partner X,which is associated with a component of a signal-generating system, isused for the purpose of quantitatively or qualitatively detecting ananalyte in a sample. In another embodiment, a further conjugateaccording to the invention which possesses specific binding sites forthe specific binding partner Y, which is associated with a component ofa signal-generating system, is additionally used for the purpose ofquantitatively or qualitatively detecting an analyte in a sample. Thenumber of binding sites for the specific binding partner X or Y shouldbe at least 2, preferably at least 5, particularly preferably at least10 and very particularly preferably at least 15. X and Y can be one andthe same specific binding partner or different specific bindingpartners. Avidin, streptavidin, an anti-digoxigenin antibody, ananti-dinitrophenol antibody, a single-stranded nucleic acid chain, ananti-hapten antibody, an enzyme, an enzyme substrate or an antibodywhich is able to bind particular polypeptides, oligopeptides or enzymesspecifically are preferably used as binding partners X or Y. Within themeaning of this invention, very particular preference is given to abinding test, preferably a homogeneous binding test, which uses one ormore of the conjugates according to the invention, with components ofthe signal-generating system being brought, as a result of the bindingof the analyte-specific binding partners, to a distance from each otherwhich permits an interaction, in particular an energy transfer, betweenthese components, and the magnitude of this interaction being measured,or with components of the signal-generating system being brought, as aresult of the binding of the analyte-specific binding partners, to adistance from each other which permits no interaction, or only veryslight interaction, in particular no energy transfer or only very slightenergy transfer, between these components, and the residual magnitude ofthis interaction being measured. In such a test, microparticles, inparticular latex particles, are preferably used as components of thesignal-generating system. Very particular preference is given to usingmicroparticle-associated photosensitizers and microparticle-associatedchemiluminescent substances as components of the signal-generatingsystem in such a test method.

It is possible to use microparticles, in particular latex particles, ascomponents of the signal-generating system in the novel methods.Microparticle-associated sensitizers, in particular photosensitizers,and microparticle-associated chemiluminescent substances are veryparticularly preferred as components of the signal-generating system.

Consequently, a microparticle, in particular a latex particle, to whicha conjugate according to the invention is bonded by way of a specificbinding partner X or Y which is bonded to the microparticle, and the useof this microparticle in a novel method, is another part of thesubject-matter of this invention. In this connection, preference isgiven to a microparticle which, as component of a signal-generatingsystem, is associated with photosensitizers or with chemiluminescentsubstances.

A test based on the LOCI method, which is described in detail in EP-0515 194, is a particularly preferred embodiment, according to theinvention, of the novel method. This test is based on usingphotosensitizers and what are termed acceptors as signal-generatingcomponents. On being exposed to light, the photosensitizers generatesinglet oxygen which reacts with the acceptors, which arechemiluminescent components. The activated chemiluminescent componentproduces light, which is measured. This preferred method will beexplained in more detail on the basis of a sandwich assay according tothe invention: the analyte is, for example, bound to an analyte-specificbinding partner R1 which can be associated with a carrier molecule whichis bound, by means of the specific binding partner X, to what are termedsensitizer particles. In the excited state, the sensitizer moleculeswhich are associated with the sensitizer particle can generate singletoxygen. This singlet oxygen can react with the chemiluminescentcompounds which are associated with what are termed chemiluminescerparticles, with the metastable compound which has been formeddecomposing once again with the generation of a light flash. Theanalyte-specific binding partner R2, which can be associated with acarrier molecule, is bound to the chemiluminescer particles by means ofthe specific binding partner Y. Since singlet oxygen is only stable fora short period in aqueous solutions, the chemiluminescer particles whichare associated with analyte-specific binding partner and which, as aresult of the formation of a sandwich complex, have arrived in theimmediate vicinity of the sensitizer particles, which have beenstimulated by light, for example, are stimulated to emit light. Thewavelength of the emitted light, which is to be measured, can be alteredusing appropriate fluorescent dyes in the chemiluminescer particles. Inthis method, the sample is preferably initially incubated with theanalyte-specific binding partners R1 and R2, after which the specificbinding partners X and Y, which are associated with the sensitizerparticles and chemiluminescer particles, respectively, are added.

Other energy transfer methods, which could also be used in the novelmethod, are based on energy transfer in accordance with Förster (Mathis,G. (1993) Clin. Chem. 39: 1953-1959; U.S. Pat. No. 5,527,684) or on theuse of photosensitizers and fluorophores (WO 95/06877) or on thecombination of radioactive irradiation and fluorophores (S. Udenfriendet al. (1985) Proc. Math. Acad. Sci. 82: 8672-8676) or on using suitableenzyme cascades (U.S. Pat. No. 4,663,278).

In another embodiment of the novel method, the analyte-specific bindingpartner R1 is associated with a component of a signal-generating systemand the analyte-specific binding partner R2 possesses specific bindingsites for the specific binding partner Y, which is associated with acomponent of a signal-generating system, with R2 possessing more thanone binding site for the given specific binding partner Y which isassociated with components of a signal-generating system.

This invention also relates to a reagent, in liquid or lyophilized form,which contains one or more of the above-described carrier moleculesaccording to the invention, which carrier molecules are in each caseassociated with one or more analyte-specific binding partners, orcontains the microparticles according to the invention. This inventionalso encompasses a test kit which contains such a reagent. This alsoapplies to the use of this reagent and/or the test kit for implementinga homogeneous or heterogeneous binding test for the purpose ofquantitatively or qualitatively detecting an analyte in a sample,preferably for implementing one of the homogeneous methods described inthe patent claims.

The invention also relates to a test kit for implementing thehomogeneous binding test according to the invention. This test kit ischaracterized by the fact that it contains an analyte-specific bindingpartner R1 which possesses more than one specific binding site for thespecific binding partner X, which is associated with a component of asignal-generating system, and by the fact that this test kit contains ananalyte-specific binding partner R2 which possesses more than onespecific binding site for the specific binding partner Y, which isassociated with a component of a signal-generating system.

The test kit according to the invention can also additionally containthe specific binding partner(s) X and/or Y which is/are associated withcomponents of a signal-generating system. In addition, the test kitsaccording to the invention can also contain a pack information leaflet,dilution buffers, standards, controls, system reagents and/or otherreagents and materials (e.g. cuvettes and sample withdrawal instruments)which are required for implementing the tests.

The test kits according to the invention preferably contain ananalyte-specific binding partner R1 which consists of one or moreanalyte-specific binding partner(s), which is/are associated withbiotinylated dextran, biotinylated protein polymers, biotinylatedantibody polymers or another carrier molecule, and/or ananalyte-specific binding partner R2 which consists of one or moreanalyte-specific binding partner(s) which is/are associated withbiotinylated dextran, biotinylated protein polymers, biotinylatedantibody polymers or another carrier molecule.

The examples which are described below serve to illustrate individualaspects of this invention and are not to be understood as representingany restriction.

EXAMPLES Example 1 Preparing Sensitizer Particles or ChemiluminescerParticles

The preparation of sensitizer particles and chemiluminescer particles isdescribed in detail in EP 0 515 194, Clin. Chem. (1996) 42: 1518-1526and Proc. Natl. Acad. Sci. (1994) 91: 5426-5430. The particles can, forexample, carry dextran envelopes and additionally possess boundstreptavidin (see also Proc. Natl. Acad. Sci. (1994) 91: 5426-5430). Thepreparation of a variant of sensitizer particles and chemiluminescerparticles is described below by way of example (see also EP 0 515 194,Example 8, for further details):

Preparing Sensitizer Particles:

A solution of chlorophyll-a in benzyl alcohol (1.0 ml; 0.6 mM) is addedto 8.0 ml of benzyl alcohol which had been heated to 105° C. Asuspension of latex beads (175 nm, carboxyl-modified latex, BangsLaboratories, Carmel, Ind.) in water (10%; 1.0 ml) is added to thebenzyl alcohol solution. The mixture is stirred at 105° C. for 5 minutesand then cooled down to room temperature. 10 ml of ethanol are added andthe mixture is centrifuged. The pellet is resuspended in a 1:1water-ethanol mixture (10 ml) and the suspension is centrifuged onceagain. The same procedure is repeated with water and the pellet issubsequently taken up in physiological sodium chloride solution.

Preparing the Chemiluminescer Particles (=Acceptor Particles):

20 ml of the carboxyl-modified latex particle suspension (10% suspensionin water) are mixed with 20 ml of 2-ethoxyethanol. The mixture is heatedto 90° C. 20 ml of a solution composed of 10 mM dioxene, 20 mM europiumchelate with the agent 3-(2-thienoyl)-1,1,1-trifluoroacetone (Kodak, CAS#14054-87-6) (EuTTA) and 60 mM trioctylphosphine oxide (TOPO) in2-ethoxyethanol are added to the particle suspension. The mixture isheated further at 97° C. for 7 minutes. After it has been cooled down toroom temperature, 40 ml of ethanol are added and the mixture iscentrifuged. The pellet is then resuspended in 80% ethanol andcentrifuged. This washing process is repeated with 10% ethanol. Inconclusion, the particles are taken up in physiological sodium chloridesolution.

Example 2 Preparing Universal Reagents

Chemiluminescer Particles (=Acceptor Particles) Containing Streptavidin:

20 mg of acceptor particles were mixed together with 2.0 mg ofstreptavidin (from Gerbu, high purity, #3058) and 0.2 mg of sodiumcyanoborohydride (from Sigma, S 8628) in a coupling buffer (0.05 Mβ-morpholinoethanesulfonic acid; from Serva, Art. 29834) and the mixturewas incubated at +37° C. for 24 hours. The coupling conditions duringthe incubation were: 50 mg of particles/ml of coupling, 0.5 mg of sodiumcyano-borohydride/ml of coupling, 2 mg of streptavidin/20 mg ofparticles.

After the incubation, 65.6 μl of a 0.48 M solution ofcarboxymethoxylamine hemihydrochloride (from Aldrich, 98% strength, Cat.No. C1,340-8) were added and mixed in and the mixture was incubated at+37° C. for a further 2 hours.

The supernatant was separated off by centrifugation and the particleswere resuspended in coupling buffer (containing 0.6 M NaCl, from Merck).After that, the supernatant was once again separated off bycentrifugation and the particles were taken up and resuspended instorage buffer (0.1 M tris-HCl, 0.3 M NaCl, 25 mM EDTA, 0.1% BSA, 0.1%dextran T-500, 0.1% zwittergent 3-14, 0.01% gentamycin, 15 ppm ofProClin-300, pH 8.0).

Sensitizer Particles Containing Streptavidin:

Sensitizer particles were coupled in analogy with the “chemiluminescerparticles (=acceptor particles) containing streptavidin” coupling.

Sensitizer Particles Containing Anti-Digoxigenin:

15 mg of sensitizer particles were mixed together with 3 mg ofanti-digoxigenin antibody (Mab DIG 2H6, from Dade Behring Inc.) and 0.15mg of sodium cyanoboro-hydride (from Sigma, S 8628) in a coupling buffer(0.05 M β-morpholinoethanesulfonic acid; from Serva, Art. 29834), andthe mixture was incubated at +37° C. for 24 hours. The couplingconditions during the incubation were: 32.8 mg of particles/ml ofcoupling, 6.6 mg of antibody/ml of coupling, 0.33 mg of sodiumcyano-borohydride/ml of coupling, 3 mg of antibody/15 mg of particles.

After the incubation, 49.2 μl of an 0.48 M solution ofcarboxymethylamine hemihydrochloride (from Aldrich, 98% strength, Cat.No. C1,340-8) were added and mixed in and the mixture was incubated at+37° C. for a further 2 hours.

The supernatant was separated off by centrifugation and the particleswere resuspended in coupling buffer (containing 0.6 M NaCl, from Merck).After that, the supernatant was once again separated off bycentrifugation and the particles were taken up and resuspended instorage buffer (0.1 M tris-HCl, 0.3 M NaCl, 25 mM EDTA, 0.1% BSA, 0.1%dextran T-500, 0.1% zwittergent 3-14, 0.01% gentamycin, 15 ppm ofProClin-300, pH 8.0).

Chemiluminescer Particles (=Acceptor Particles) ContainingAnti-Digoxigenin or Anti-Troponin:

Acceptor particles were coupled to antibody directed against digoxigeninor against troponin in analogy with the “sensitizer particles containinganti-digoxigenin” coupling.

In the following examples, universal reagent A (acceptor particlescontaining streptavidin) is used in combination with universal reagent B(sensitizer particles containing anti-digoxigenin), or universal reagentA (acceptor particles containing anti-digoxigenin) is used incombination with universal reagent B (sensitizer particles containingstreptavidin).

Example 3 PSA Assay

Preparing PSA Specific Reagents

Conjugate C1 (Biotinylated Anti-PSA Antibody):

0.23 mg of biotin-LC-NHS (from Pierce, Art. 21336, Immuno Pure),dissolved in DMSO (from RdH, 34943), was added to 2.3 mg of anti-PSAantibody (MAK <PSA> 92-284/03, Dade Behring Marburg GmbH, in 0.1 Msodium carbonate (from RdH, 31432)) and mixed in, and the mixture wasincubated at +4° C. for 16 hours. Molar ratio [Ab]: [biotin-LC-NHS]employed=1:35.

The conjugate was purified through a PD-10 Sephadex G-25M (fromPharmacia Biotech, Code 17-0851-01) column using phosphate buffer (0.05M sodium dihydrogen phosphate containing 0.15 M sodium chloride, pH7.5).

Conjugate C2 (Digoxigenin-Labeled Anti-PSA Antibody):

The anti-PSA antibody (MAK <PSA> 92-283/029 Dade Behring Marburg GmbH)was labeled with digoxigenin in accordance with the DIG-antibodylabeling kit (from Boehringer Mannheim Biochemica, Order No. 1367200,implementation: protein labeling with DIG-NHS 1. monoclonal antibodies)directions/instructions for use.

Assay Buffer

0.1 mol of tris/l plus 0.3 mol of NaCl/l plus 25 mmol of EDTA/l plus0.1% RSA plus 0.1% dextran T-500 plus 0.1% zwittergent 3-14 plus 0.01%gentamycin plus 15 ppm of ProClin-300, pH 8.00.

Implementing a PSA Assay

In order to carry out the test, the components were mixed and incubatedas follows:

10 μl of sample 75 μl of assay buffer 25 μl of conjugate C1(biotinylated anti-PSA antibody) and conjugate C2 (digoxigeninatedanti-PSA antibody), in each case 0.96 μg/ml 371   seconds of incubationat +37° C. 100 μl of assay buffer 20 μl of sensitizer particlescontaining anti-digoxigenin (0.2 mg/ml) 20 μl of acceptor particlescontaining streptavidin (0.2 mg/ml) 762.5 seconds of incubation at +37°C.Measurement

The test was carried out and measured on a modified Tecan SampleProcessor, see Ullman et al. (Clinical Chemistry 42: 1518-1526, 1996, EP0515194 A2), and the signals were recorded.

Results

TABLE 1 Standard curve in the PSA assay: Signal PSA standard No. ng/ml[counts] 1 0 2810 2 0.1 3031 3 0.3 3679 4 0.9 4611 5 3.9 10316 6 1024178 7 34.8 114241 8 79.4 326876 9 270 856074

TABLE 2 Samples which were measured using the PSA assay according to theinvention: Reference test: Abbott-IMx PSA assay total PSA PSA assay PSAPSA Sample ID Signal concentration concentration (PSA sera) [counts][ng/ml] [ng/ml] OF 22 6171.5 2.2 2.02 OF 23 5204.0 1.5 1.11 OF 2410443.0 4.4 3.65 OF 25 3301.0 0.1 0.22 OF 27 35440.0 13.7 12.41 OF 2812248.5 5.2 4.79 OF 29 28453.0 11.4 9.94 OF 30 95165.5 30.2 29.26 OF 315012.0 1.4 1.09 OF 32 12934.5 5.5 5.37 OF 33 4959.0 1.4 1.00 OF 3550551.5 18.3 18.15 OF 40 4737.0 1.2 0.61 OF 45 4196.0 0.8 0.48 OF 5022371.0 9.2 9.89 OF 55 3989.5 0.7 0.67 OF 60 5869.5 2.0 1.86

The above table shows that the values determined in the novel methodagreed, within normal limits, with those in the comparison method(Abbott IMx total PSA, list 5 No. 1D85). This verifies that the methodaccording to the invention functions.

Example 4 Varying the Number of Biotin Labels Per Antibody

The anti-PSA antibody (Mab<PSA>92-284/03, Dade Behring Marburg GmbH) wasconjugated with varying quantities of biotin molecules, and the anti-PSAantibody (Mab<PSA>92-283/09, Dade Behring Marburg GmbH) was conjugatedwith varying quantities of digoxigenin molecules, in accordance with thebiotinylation method described in Example 3. The results obtained withthe antibody pairs are shown in the following table.

TABLE 3 PSA assay based on antibodies having differing numbers ofbinding sites for the specific binding partners X and Y. Measurementsignal given in counts. Cali- 1 biotin 2 biotin or 3 biotin or 5 biotinor brator or digoxi- digoxigenin digoxigenin digoxigenin nominal geninper molecules molecules molecules value antibody per antibody perantibody per antibody [ng/ml] [counts] [counts] [counts] [counts] 0 26742600 2461 2542 0.1 8622 7055 6019 4704 0.3 13080 11492 9839 8334 1.018442 21248 21599 21963 3.0 31959 52192 62751 71571 10.0 101813 191158251553 278551 30.0 345151 656275 812705 953906 50.0 578619 10161001227390 1438690 100.0 1258490 1983200 2339990 2593270

Example 5 Preparing Dextran-Antibody-Biotin Conjugates

Activating Antibodies with N-succinimidyl S-acetylthio-acetate (SATA)

10.4 mg of anti-PSA antibody (Mab<PSA>92-284/03, Dade Behring MarburgGmbH) in 0.1 M sodium carbonate buffer (from Riedel de Haen, 31432) arerebuffered in mixed buffer (LiBO₃/20% dioxane)., pH 8.5, and mixed with104 μl of SATA (from Pierce) in DMF (2 mg/ml). After the mixture hadbeen incubated at 37° C. for 1.5 hours, it is incubated for 45 minuteswith 200 μl of NH₂OH.

The conjugate is then desalted through a PD-10 column using 0.1 Mphosphate buffer, pH 6.0.

Preparing Activated Biotin-Dextran

3.9 mg (1.95 ml) of FlukaBioDex (70000 kDa, product number 14402, biotinsubstitution 20 mol/mol) are taken up in mixed buffer (2 mg/ml) andmixed with 26 μl of GMBS solution (N-maleimidobutyryloxysuccinimideester, from Pierce) in dioxane (6 mg/ml). This mixture is incubated at18° C. for 1 hour. The conjugate is then desalted through a PD-10 columnusing 0.1 M phosphate buffer, pH 6.0.

Coupling the Activated Antibody to the Activated Biotin-Dextran

Conjugate 1:

1.7 ml of the antibody-SATA solution (1 mg/ml) are mixed with 509 μl ofthe FlukaBioDex-GMBS solution (1.32 mg/ml). This mixture is incubated at37° C. for 2 hours and then stopped with 220 μl of an 0.1 M solution ofn-ethylmaleimide. The purification/desalting takes place throughSephacryl S300 (diameter 1.6 cm, gel bed height 90 cm, quantity loadedapprox. 2 ml) using 0.1 M TRIS/HCl, 150 mM NaCl, pH 7.4. The fractionscontaining the conjugate were pooled and concentrated down to 1.7 ml(≈0.65 mg/ml).

Conjugate 2:

1.7 ml of the antibody-SATA solution (1 mg/ml) are mixed with 102 μl ofthe FlukaBioDex-GMBS solution (1.32 mg/ml). This mixture is incubated at37° C. for 2 hours and then stopped with 180 μl of an 0.1 M solution ofn-ethylmaleimide. The purification/desalting takes place through FlukaSephacryl S300 (diameter 1.6 cm, gel bed height 90 cm, quantity loadedapprox. 2 ml) using 0.1 M TRIS/HCl, 150 mM NaCl, pH 7.4. The fractionscontaining the conjugate were pooled and concentrated down to 1.7 ml(≈0.64 mg/ml).

Because of the different formulations, a ratio of approx. 18 biotinmolecules per antibody was obtained for conjugate 1 and a ratio ofapprox. 3 biotin molecules per antibody is obtained for conjugate 2.

These conjugates were used in the above-described PSA assay incombination with the above-described digoxigeninated anti-PSA antibodieshaving increasing antibody/digoxigenin ratios. The concentration of thedextran conjugates was 9.75 μg/ml and the digoxigeninated anti-PSAantibodies were used at a concentration of 8.8 μg/ml. The results areshown in the following table.

TABLE 4 PSA assay using dextran-antibody-biotin conjugates. Measurementsignal given in counts. Conjugate 1 Conjugate 1 Conjugate 2 Conjugate 2(18 biotin molecules (18 biotin molecules (3 biotin molecules (3 biotinmolecules per antibody) in per antibody) in per antibody) in perantibody) in combination with combination with combination withcombination with digoxigeninated digoxigeninated digoxigeninateddigoxigeninated Calibrator anti-PSA antibody anti-PSA antibody anti-PSAantibody anti-PSA antibody nominal value (Ab/Dig 1:1) (Ab/Dig 1:5)(Ab/Dig 1:1) (Ab/Dig 1:5) [ng/ml] [counts] [counts] [counts] [counts] 04309 4157 4937 4981 0.1 6745 11647 5826 7613 0.3 11837 27686 7817 134161.0 32488 84783 15512 33460 3.0 116736 287554 44103 98896 10.0 4694061108220 152400 375343 30.0 1655420 3176980 1060780 1259030 50.0 27128704573200 2501150 2076760 100.0 4957770 7049890 2339990 4166780

Example 6 Rubella IgM Assay

Preparing Rubella IgM-Specific Reagents

Conjugate C1 (Biotinylated Anti-Human IgM Antibody):

Goat anti-human IgM antibody (Pab <h-IgM> 62FX022, Dade Behring MarburgGmbH) was biotinylated in analogy with the biotinylation of the anti-PSAantibodies (see Example 3).

Conjugate C2 (Digoxigenin-Labeled Anti-Rubella Anti-Body):

The anti-rubella antibody (Mab <rubella> 93-9/08, Dade Behring MarburgGmbH) was labeled with digoxigenin in accordance with the DIG-antibodylabeling kit (from Boehringer Mannheim Biochemica, order No. 1367200,implementation: protein labeling using DIG-NHS 1. monoclonal antibodies)directions/instructions for use.

Assay Buffer:

0.1 mol of tris/l plus 0.3 mol of NaCl/l plus 25 mmol of EDTA/l plus0.1% RSA plus 0.1% dextran T-500 plus 0.1% zwittergent 3-14 plus 0.01%gentamycin plus 15 ppm of ProClin-300, pH 7.3.

Rheumatoid Factor (RF) Absorbent:

Rheumatoid factor (RF) absorbent from Dade Behring Marburg GmbH, productnumber OUCG.

Rubella Antigen:

From Intergen, CDP (lot 8320)

Implementing a Rubella IgM Assay

In order to carry out the test, the components were mixed and incubatedas follows:

10 μl of sample, diluted 1 + 9 parts in RF absorbent 40 μl of assaybuffer 25 μl of conjugate C1 (biotinylated anti-human IgM antibody, 8μg/ml) and conjugate C2 (digoxigeninated anti- rubella antibody, 2μg/ml) 25 μl of rubella antigen (4 [μg/ml) 453.5 seconds of incubationat +37° C. 75 μl of assay buffer 25 μl of acceptor particles containinganti-digoxigenin (0.05 mg/ml) 50 μl of sensitizer particle containingstreptavidin (0.4 mg/ml) 432.5 seconds of incubation at +37° C.

Measurement

The test was carried out and measured on a modified Tecan SampleProcessor, see Ullman et al. (Clinical Chemistry 42: 1518-1526, 1996, EP0515 194 A2), and the signals were recorded.

Result of a Rubella IgM Assay

TABLE 5 Measuring samples in the rubella IgM assay. The measurementsignal is given in counts or in mE (extinction measurement). DiaSorinRubella IgM reference test DiaSorin Sample ID [counts] [mE] assessment665550 4383 101 negative NS24 13477 452 borderline 6DD416 22371 1825positive 35-033 131152 >measurement positive range

As is evident from the above table, the results which were obtainedusing the method according to the invention agree, within normal limits,with those obtained in the comparison method (DiaSorin, ETI-RUBEK-Mreverse, P2471). This verifies that the method according to theinvention also functions in an indirect test procedure.

Example 7 Using a Troponin Assay to Compare a Biotin Standard Conjugatewith a Biotinylated Carrier Molecule-Fab′ Conjugate

Preparing a Biotinylated Carrier Molecule-Fab′ Conjugate (“M-IgG-BiotinAnti-Troponin Conjugate”):

A solution of murine IgG antibodies (“M-IgG”) (15.0 ml, 2.6 mg ofM-IgG/ml, 0.26 mmol) in 0.1 M phosphate buffer (5 mM EDTA, pH 7.0) ismixed with an aqueous solution of sulfosuccinimidyl(4-iodoacetyl)amino-benzoate (0.66 ml, 2.0 mg/ml). After a reaction timeof 1 hour at 25° C., the mixture is concentrated using a gel filtrationcolumn (AcA22, Ciphergen, Fermont, Calif.), and purified. The fractionscontaining the monomeric, activated M-IgG (HPLC-tested) are pooled.

A solution (3.0 ml, 10 mg/ml) of an F(ab′)₂ fragment of an anti-troponinantibody in an 0.1 M phosphate buffer (5 mM EDTA, pH 6.0) is mixed with0.091 ml of a mixture of dithiothreitol (15.4 mg/ml) and2-mercaptoethanol (15.6 μl/ml). After a reaction time of 1 hour at 37°C., the mixture was concentrated using a gel filtration column (AcA22,Ciphergen, Fermont, Calif.), and purified. The fractions containing theFab′ antibody fragment are pooled.

A mixture of the activated M-IgG (20.5 mg; 0.14 mmol) and the Fab′fragment (20.5 mg; 0.41 mmol) is rebuffered into a phosphate buffer pH7.0 (5 mM EDTA) using an Amicon ultrafiltration cell. The mixture isconcentrated down to 5.0 mg of protein/ml and incubated at 2-8° C. for24-70 hours. After that, an aqueous solution of N-ethylmaleimide (20mg/ml; 50 μl per ml of protein solution) is added. After one hour atroom temperature, the solution is concentrated in 10 mM phosphate buffer(300 mM NaCl, pH 7.0) using a gel filtration column (AcA22, Ciphergen,Fermont, Calif.), and purified. The protein fractions are pooled.

An aqueous solution of NHS-PEO4-Biotin (Pierce Chemical Company,Rockford, Ill.; 0.095 ml, 0.5 mg/ml) is added to the Fab′-M-IgGconjugate in the pH 7.0 phosphate buffer (6 ml; 0.8 mg/ml; 16 μmol).After a reaction time of 4 hours at room temperature, the mixture isdiafiltered against the pH 7.0 phosphate buffer.

Preparing an Fab′-Biotin Conjugate (“Standard Biotin Anti-TroponinConjugate”)

A solution of the Fab′ fragment of the anti-troponin antibody (2 ml of a5 mg/ml protein solution) in 10 mM phosphate buffer (300 mM NaCl, pH7.0) is mixed with 0.22 ml of a PEO-iodoacetylbiotin solution (10 mg/ml;4 mmol in DMF) from Pierce Chemical Company, Rockford, Ill. After areaction time of 4 hours at room temperature, the mixture is diafilteredagainst the pH 7.0 phosphate buffer. The protein concentration wasdetermined using the BCA protein assay supplied by Pierce ChemicalCompany.

Troponin Immunoassays

A) Test Implementation

In order to carry out the test, the components were mixed and incubatedas follows:

20 μl of sample 10 μl of water 15 μl of conjugate (standard biotinanti-troponin conjugate: 12.5 μg/ml or M-IgG-biotin anti-troponinconjugate: 8 μg/ml) 13 μl of acceptor particles containing anti-troponinantibody (210 μg/ml) 435 seconds of incubation at +37° C. 13 μl ofsensitizer particles containing streptavidin (1.5 mg/ml) 10 μl of water 87 seconds of incubation at +37° C. 169 μl of water

Measurement

The test was carried out and measured on a modified Tecan SampleProcessor, see Ullman et al. (Clinical Chemistry 42: 1518-1526, 1996, EP0515194 A2), and the signals were recorded.

B) Results

TABLE 6 Troponin immunoassay using two different anti-troponinconjugates. Measurement signal given in counts. Troponin Standard biotinM-IgG-biotin calibrator anti-troponin anti-troponin nominal valueconjugate conjugate [ng/ml] [counts] [counts] 0 5236 4337 0.025 60087475 0.05 6980 10684 0.1 8744 17421 0.5 22752 73511 2.25 91614 362965

The biotinylated carrier molecule-Fab′ conjugate (=M-IgG-biotinanti-troponin conjugate) gives a much steeper calibration curve, therebyimparting greater precision to the test.

1. A homogeneous method for quantitatively or qualitatively detecting ananalyte in a sample (sample analyte) comprising (1) contacting thesample with (a) a first conjugate molecule comprising ananalyte-specific binding partner R1 and possessing specific bindingsites for a specific binding partner X, wherein R1 is capable of bindingto the analyte or to an analyte-specific binding partner R2; (b) asecond conjugate molecule comprising an analyte-specific binding partnerR2 and possessing specific binding sites for a specific binding partnerY, wherein R2 is capable of binding to the analyte or to R1; (c) thespecific binding partner X, which is associated with a first componentof a signal-generating system; and (d) the specific binding partner Y,which is associated with a second component of the signal-generatingsystem; wherein at least one of the conjugates comprising R1 or R2possesses more than one binding site for the respective specific bindingpartner X or Y; wherein the first and second components of thesignal-generating system enter into a detectable interaction whenspatially close to each other and the detectable interaction comprises adirect energy transfer between the components; wherein (i) both R1 andR2 are able to bind specifically to the sample analyte, or (ii) only R1is able to bind specifically to the sample analyte and R2 can competewith the sample analyte in binding to R1; and (2) detecting the analytein the sample (sample analyte) by measuring the magnitude of theinteraction between the components of the signal-generating system;wherein said method is a homogeneous binding test.
 2. The method ofclaim 1, wherein R1 is bound to the component of the signal-generatingsystem by way of X during or after the binding reaction with theanalyte.
 3. The method of claim 1, wherein the number of binding sitespossessed by the conjugate comprising R1 for the specific bindingpartner X is at least
 2. 4. The method of claim 1, wherein the number ofbinding sites possessed by the conjugate comprising R2 for the specificbinding partner Y is at least
 2. 5. The method of claim 1, wherein R1and R2 are the same analyte-specific binding partner.
 6. The method ofclaim 1, wherein R1 and R2 are able to bind specifically to the sampleanalyte.
 7. The method of claim 1, wherein the binding sites possessedby the conjugate comprising R1 for the specific binding partner X arechosen from hapten, antigen, biotin, digoxigenin, fluorescein,single-stranded nucleic acid chain, and dinitrophenol.
 8. The method ofclaim 1, wherein the binding sites possessed by the conjugate comprisingR2 for the specific binding partner Y are chosen from hapten, antigen,biotin, digoxigenin, fluorescein, single-stranded nucleic acid chain,and dinitrophenol.
 9. The method of claim 1, wherein X and Y are thesame specific binding partner.
 10. The method of claim 1, wherein X isavidin, streptavidin, an anti-digoxigenin antibody, ananti-dinitrophenol antibody, a single-stranded nucleic acid chain, ananti-hapten antibody, an enzyme, or an enzyme substrate, or an antibodywhich specifically binds to a peptide antigen.
 11. The method of claim1, wherein Y is avidin, streptavidin, an anti-digoxigenin antibody, ananti-dinitrophenol antibody, a single-stranded nucleic acid chain, ananti-hapten antibody, an enzyme, or an enzyme substrate, or an antibodywhich specifically binds to a peptide antigen.
 12. The method of claim1, wherein the components of the signal-generating system are brought toa distance from each other which permits a detectable interactionbetween these components, as a result of the analyte being bound to atleast one of R1 and R2.
 13. The method of claim 1, wherein thecomponents of the signal-generating system are not brought to a distancefrom each other which permits a detectable interaction between thesecomponents, as a result of R1 being bound by sample analyte incompetition with R2.
 14. The method of claim 1, wherein the componentsof the signal-generating system are microparticles associated withphotosensitizers or microparticles associated with chemiluminescentsubstances.
 15. The method of claim 1, wherein R2 is bound to componentsof the signal-generating system by way of Y during or after the bindingreaction with the analyte.
 16. The method of claim 3, wherein the numberof binding sites possessed by the conjugate comprising R1 for thespecific binding partner X is at least
 5. 17. The method of claim 3,wherein the number of binding sites possessed by the conjugatecomprising R1 for the specific binding partner X is at least
 10. 18. Themethod of claim 3, wherein the number of binding sites possessed by theconjugate comprising R1 for the specific binding partner X is at least15.
 19. The method of claim 4, wherein the number of binding sitespossessed by the conjugate comprising R2 for the specific bindingpartner Y is at least
 5. 20. The method of claim 4, wherein the numberof binding sites possessed by the conjugate comprising R2 for thespecific binding partner Y is at least
 10. 21. The method of claim 4,wherein the number of binding sites possessed by the conjugatecomprising R2 for the specific binding partner Y is at least
 15. 22. Themethod of claim 14, wherein the microparticles are latex particles. 23.The method of claim 1, wherein the first component of thesignal-generating system is an energy donor and the second component ofthe signal-generating system is an energy recipient.
 24. The method ofclaim 1, wherein one component of the signal-generating system is aphotosensitizer.
 25. The method of claim 1, wherein at least onecomponent of the signal-generating system is a fluorophore.
 26. Themethod of claim 1, wherein at least one of the analyte-specific bindingpartners R1 and R2 is an antibody.
 27. The method of claim 1, whereinboth analyte-specific binding partners R1 and R2 are antibodies.
 28. Themethod of claim 1, wherein the first and second conjugates furthercomprise a carrier molecule to which are bound the analyte-specificbinding partners R1 and R2 and the specific binding sites for thespecific binding partners X and Y.
 29. The method of claim 1, whereinthe specific binding partners X and Y are bound to microparticles thatalso comprise the components of the signal-generating system.